Hepatoprotective Effect of Gallotannin‑enriched Extract Isolated from Gall on Hydrogen Peroxide‑induced Cytotoxicity in HepG2 Cells

Background: Gall (Galla Rhois [GR]) is known to have antibacterial, anti‑inflammatory, antimetastatic, and anti‑invasion activities and exert hepatoprotective effects. However, the hepatoprotective effects of gallotannin‑enriched GR (GEGR) and their mechanisms have not yet been investigated. Objective: The potential protective effect of GEGR against hepatotoxicity induced by hydrogen peroxide (H2O2) was investigated. Materials and Methods: Changes in cell viability, apoptosis protein expression, and reactive oxygen species (ROS) generation were determined in HepG2 cells that were pretreated with four different concentrations of GEGR (6.25–50 µg/ml) for 24 h before H2O2 exposure. Results: GEGR consisted of gallotannin (69.2%), gallic acid (26.6%), and methyl gallate (4.2%) and showed remarkable 2,2‑diphenyl‑1‑picrylhydrazyl scavenging activity (inhibitory concentration 50% = 0.212 µg/ml). The lethal dose 50% and effective dose 50% values for the response of HepG2 cells to GEGR were determined to be 178 and 6.85 µg/ml, respectively. Significant reductions in the immunofluorescence intensity indicating apoptosis were also detected in the nuclei of HepG2 cells stained with 4’,6‑diamidino‑2‑phenylindole and Annexin V after GEGR treatment. The Bax/Bcl‑2 ratio and active caspase‑3 level were higher in H2O2 + vehicle‑treated cells. However, these levels gradually decreased to those of the No‑treated group in the GEGR pretreated group even though little or no decrease was observed in response to low GEGR concentrations. Furthermore, the GEGR pretreated group showed a reduced level of 2′,7′‑dichlorofluorescein diacetate stained cells, indicating ROS generation relative to the H2O2 + vehicle‑treated group. Conclusion: The results of this study provide strong evidence that GEGR can prevent cell death induced by H2O2 in HepG2 cells through the induction of antioxidant conditions.